Reaction with antibodies

A second method of immunotoxin preparation by reductive amination involves the use a polysaccharide spacer. Soluble dextran may be oxidized with periodate to form a multifunctional crosslinking polymer. Reaction with antibodies and cytotoxic molecules in the presence of a reducing agent forms multivalent immunotoxin conjugates. The following sections discuss these options. 

W. M. Hunter, The Preparation of Radioiodinated Proteins of High Activity, Their Reaction With Antibody in Vitro the Radioimmunoassay, in Handbook ofExperimen -tal Immunology (D. M. Weir, Ed.), F. A. Davis, Philadelphia, 1967. 

A frequently encountered problem when performing immunoblotting is to make a direct correlation between the total electrophoretic pattern and the bands detected by reaction with antibodies. A number of stains, such as amido black, India ink, and colloidal gold, have been described for detection of proteins on nitrocellulose (1-3). When these stains are used, a duplicate nitrocellulose blot has to be made, which may lead to discrepancies when comparing two patterns. The only stain that binds reversibly to proteins and does not give high background on nitrocellulose is Ponceau S (4). The detection limit of proteins stained with Ponceau S is between 250-500 ng. 

After reaction with antibody the immunoadsorbent is washed with buffer six times to remove non-antibody protein. The success of the reaction is then determined by measuring the total protein present and comparing it with the protein on the unreacted immunoadsorbent. Antibody uptake is assumed to be the difference between these two figures. 

The enzyme activity is often not abrogated by a reaction with antibodies. Thus, an enzyme-anti-enzyme complex can be introduced by an anti-Ig into the complex and eliminates the inactivation of the enzyme or of the antibody by the chemical linkage, reduces background staining caused by the introduction of chemically reactive groups and, at the same time, enhances detectability. 

After autoclaving in TB containing 200 mM NaCl for 10 min, the sections were further treated with TB for 15 min at 95-98°C. All antibodies partially recovered their immunostaining after the second heating (Table 17.3). These results demonstrate that the ionic strength of the solution is one of the critical factors for HIAR, and that a high concentration of salt inhibits the exposure of epitopes, preventing their reaction with antibodies. 

Enzymatic detection Reaction with antibody anti-FlTC AP conjugate (Ab-AP) an aliquot of 40 /u,L of Ab-AP solution (1/100 dilution) is dropped on the genosensor device for 60 minutes. Then a washing step with 0.1 M Tris buffer pH 9.8, containing 1% BSA, is carried out. 

Using the Method 2 assay for fliioredoxin m, we have reanalyzed the extracts from y-1 cells grown in the dark and have found some thioredoxin m activity in Ae extracts (Table 1). Western blot analysis of these samples, however, showed no significant cross reaction with antibodies to thioredoxin m. 

Shields RL, Werther WR, Zioncheck K, O Connell L, Fendly B, Presta LG, Thomas D, Saban R, Jardieu PM. Inhibition of allergic reactions with antibodies to IgE. Int Arch Allergy Immunol 1995 107 308-312. 

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